Latest revision as of 13:14, 2 October 2024

iaaM-iaaH

The iaaM and iaaH genes jointly participate in the biosynthesis pathway of the plant hormone Indole-3-acetic acid (IAA). Firstly, it is converted into IAM through tryptophan-2-monooxygenase (IaaM) encoded by the iaaM gene. In the second step, IAM is converted into IAA by IAM hydrolase (IaaH), which is encoded by the iaaH gene.

Usage and Biology:

BBa_K5468006 encodes two critical enzymes, tryptophan-2-monooxygenase (iaaM) and indole-3-acetamide (IAM) hydrolase (iaaH), which are involved in the biosynthesis of indole-3-acetic acid (IAA), a crucial plant hormone known as auxin. Auxins regulate plant growth and development, particularly promoting cell elongation, root formation, and differentiation. This part enables the production of IAA in engineered microorganisms, especially Escherichia coli.

Biological Function:

1、Tryptophan Conversion to IAM (iaaM):The iaaM gene encodes tryptophan-2-monooxygenase, which catalyzes the first step of IAA biosynthesis via the IAM pathway. It converts L-tryptophan, an essential amino acid, into indole-3-acetamide (IAM). This reaction is crucial for initiating the IAM pathway, providing the intermediate necessary for further conversion into IAA.

2、IAM to IAA Conversion (iaaH):The iaaH gene encodes IAM hydrolase, which catalyzes the hydrolysis of indole-3-acetamide into indole-3-acetic acid (IAA). This final step in the pathway is essential for producing biologically active IAA, which is responsible for a wide range of growth-promoting activities in plants.

3、Polycistronic Design and Expression:The iaaM and iaaH genes are arranged in a polycistronic structure, meaning both genes are transcribed together under a single promoter. This ensures coordinated expression of both enzymes, optimizing the efficiency of the IAA production pathway. The inclusion of B0034 ribosome binding sites between the two genes ensures robust translation in bacterial systems such as E. coli. The genes are cloned into the pET23b vector, which allows high-level expression under the control of the T7 promoter, commonly used in BL21(DE3) strains of E. coli.

Figure. 1. Schematic diagram of IAM Pathway

Applications:

IAA Production in Engineered Strains:The primary use of this part is to engineer bacterial strains that can produce IAA. This is valuable for both basic research into auxin biology and for practical applications such as promoting plant growth or modulating plant responses. For example, when transformed into E. coli BL21, the strain can produce significant amounts of IAA, which can be tested for its biological activity on plant tissues.

Auxin-mediated Plant Growth Enhancement:IAA is known to stimulate root growth, cell differentiation, and shoot elongation. This part can be utilized in experiments designed to enhance these growth responses. Engineered bacteria producing IAA could be applied to plant rhizospheres to enhance root development, especially under stressful conditions such as low nutrient availability or soil contamination (e.g., petroleum stress).

Environmental and Agricultural Applications:The engineered bacterial strains expressing BBa_K5468006 could potentially be used in bioremediation to promote plant growth in contaminated soils. In experiments, IAA produced by the bacteria has been shown to improve root development under petroleum stress. This indicates that the part could be used in agricultural or environmental settings where improving plant resilience to harsh conditions is critical.

Characterization：

1.Construction of IAA-Producing Engineered Strains •Construction of engineered strains containing iaaM and iaaH genes for IAA production via the IAM pathway.

2.Construction of the IAA Standard Curve •Using the Salkowski reagent method to measure the absorption at 530 nm for different concentrations of IAA and create a standard curve.

3.Measurement of IAA Production •Testing whether the engineered strain BL21-pET23b-iaaM-iaaH produces significant amounts of IAA.

4.Time-Course Analysis of IAA Production •Analyzing the time-dependent production of IAA in the engineered strain to determine the optimal production time point.

5.Effect of Light on IAA Production •Investigating the effect of light on IAA production and determining the best cultivation conditions (in the dark).

6.Effect of Engineered Strain Supernatant on Seed Germination •Testing the influence of supernatant from IAA-producing strains on the germination rates of various seeds (water spinach, soybeans, wheat, black wheat).

7.Effect of Engineered Strain Supernatant on Root Growth Under Petroleum Stress •Investigating the effect of IAA-containing supernatant on root growth of water spinach and soybean seeds under petroleum-contaminated conditions.

Construction of IAA-Producing Engineered Strains via the IAM Pathway

Objective and Methods

In this experiment, the goal was to construct an engineered strain that produces indole-3-acetic acid (IAA) via the IAM pathway. The biosynthesis begins with tryptophan, which is first converted to indole-3-acetamide (IAM) by tryptophan-2-monooxygenase, encoded by the iaaM gene. In the second step, IAM is hydrolyzed to IAA by IAM hydrolase, encoded by the iaaH gene. The synthesized iaaM and iaaH genes, arranged in a polycistronic structure with ribosome binding sites (RBS B0034) between the genes, were cloned into the pET23b vector (Azenta, USA) using NdeI and XhoI restriction sites. After sequencing verification, the recombinant plasmids were transformed into E. coli BL21 for expression.

Figure 2. Gel Electrophoresis of IaaM and IaaH Gene Constructs for IAA Production

Results and Conclusion

The gel electrophoresis results shown above demonstrate successful amplification of the iaaM and iaaH genes, with bands at approximately 1635 bp and 1341 bp, respectively, confirming the correct size of the inserted genes. This indicates that the genes were successfully cloned into the vector, laying the foundation for the production of IAA via the IAM pathway.

Construction of the IAA Test Standard Curve

Objective and Methods

To construct the IAA test standard curve, various concentrations of IAA were dissolved in PBS (pH 7.4). For each sample, 1 mL of IAA solution was mixed with 1 mL of Salkowski reagent. The Salkowski reagent contains 0.5% FeCl₃ in 35% perchloric acid. The mixtures were incubated in the dark for 30 minutes. Reaction Principle: In a strongly acidic environment created by sulfuric acid, IAA is oxidized to form an indole derivative with a conjugated double bond. This derivative reacts with iron ions (Fe³⁺) to form a colored complex, turning the solution pink or purple. The intensity of the color correlates with the concentration of IAA, which can be measured by detecting the absorption value at 530 nm.

Figure 3. IAA Standard Curve Using Salkowski Reagent

Results and Conclusion

Using spectrophotometry, the absorbance at 530 nm was measured for different concentrations of IAA, resulting in the standard curve shown above. The linear regression equation derived from the data is Y = 0.005299X + 0.05417, with an R² value of 0.98, indicating a strong linear relationship between IAA concentration and absorbanc. This standard curve enables accurate quantification of IAA in experimental samples.

Production of IAA by Engineered Strains via the IAM Pathway

Objective and Methods

To test whether the engineered strain BL21-pET23b- iaaM- iaaH can produce significant amounts of indole-3-acetic acid (IAA), the following experiment was conducted. The bacteria were first grown overnight in LB medium at 37°C with shaking at 180 rpm. The next day, 1 mL of bacterial culture was transferred to 50 mL of fresh LB medium and incubated under the same conditions. To prevent light-induced degradation of IAA, the flasks were covered with aluminum foil. After 12 hours of incubation, 1.5 mL of bacterial culture was centrifuged at 10,000 rpm for 1 minute. The supernatant (1 mL) was collected and mixed with 1 mL of Salkowski reagent. The mixture was incubated in the dark for 30 minutes, and the absorbance at 530 nm was measured to determine IAA concentration based on the standard curve.

Figure 4. IAA Production by Engineered Strains via IAM Pathway

Results and Conclusion

The results, as shown in the accompanying figure, demonstrate that the engineered strain BL21-pET23b- iaaM- iaaH produced the highest concentration of IAA after 12 hours at 37°C, significantly higher than the control strains BL21 and BL21-pET23b. This confirms that the iaaM and iaaH genes in the engineered strain successfully enabled the production of IAA. Therefore, the BL21-pET23b- iaaM- iaaH strain is the most effective choice for auxin production. This confirms the strain's capability to synthesize IAA through the IAM pathway, making it a strong candidate for applications requiring auxin production.

Time-Course Analysis of IAA Production by Engineered Strains via the IAM Pathway

Objective and Methods

To analyze the time-dependent production of IAA by the engineered strain BL21-pET23b- iaaM- iaaH, the bacteria were grown overnight in LB medium at 37°C and 180 rpm. The following day, 1 mL of bacterial culture was transferred to 50 mL of fresh medium and incubated under the same conditions. Samples (1 mL) were taken at 0, 4, 6, 8, 12, and 20 hours. After centrifugation, the supernatant was collected, and the IAA concentration was determined using the Salkowski method.

Figure 5. Time-Course of IAA Production by IAM Pathway Strains

Results and Conclusion

As shown in the time-course curve, IAA production increased rapidly and reached its maximum rate at 8 hours, peaking at 15 hours before stabilizing. Based on these results, a cultivation period of 15 hours was chosen as optimal for future experiments involving seed treatment.

Effect of Light on IAA Production in IAM Pathway Engineered Strains

Objective and Methods

To determine whether IAA is degraded by light, the engineered strain BL21-pET23b- iaaM- iaaH was grown overnight in LB medium at 37°C and 180 rpm. The bacterial culture was then divided into two groups: one covered with aluminum foil (dark condition), and the other exposed to light (light condition). Both groups were incubated for 12 hours under the same conditions. After incubation, 1 mL of culture was collected, centrifuged, and the supernatant was analyzed for IAA concentration using the Salkowski method.

Figure 6. Effect of Light on IAA Production

Results and Conclusion

The results, shown in the accompanying figure, demonstrate that IAA production in the light-exposed group was nearly zero, while the dark-incubated group produced normal levels of IAA. This indicates that IAA is highly sensitive to light and is degraded when exposed. Therefore, cultivation of IAA-producing strains should be conducted in the dark to prevent auxin degradation.

Effect of Engineered Strain Supernatant on Seed Germination

Objective and Methods

To evaluate the influence of the supernatant from engineered strains on seed germination, four types of seeds were selected: water spinach, soybeans, wheat, and black wheat. For each type, 200 seeds were divided into an experimental group and a control group, with 100 seeds in each. The experimental group was treated with the supernatant from IAM pathway-engineered strains, while the control group was treated with water. The engineered strain was cultured overnight in LB medium at 37°C and 180 rpm. The following day, 1 mL of the bacterial culture was transferred to 50 mL of fresh medium, wrapped in aluminum foil to avoid light degradation. After 12 hours of incubation, the culture was centrifuged to collect the supernatant. Seeds in the experimental group were soaked in this supernatant for 12 hours, while control seeds were soaked in water. After soaking, the seeds were transferred to germination trays with water. Seed germination was observed after two days.

Figure 7. Seed Germination Results with IAM Pathway Strain Supernatant

Results and Conclusion

 Soybeans: Control group germination rate 68%, experimental group 91%.
 Wheat: Control group 59%, experimental group 83%.
 Water spinach: Control group 53%, experimental group 78%.
 Black wheat: Control group 64%, experimental group 87%.

The images above compare the control and experimental groups, demonstrating a notable improvement in seed germination rates when treated with the supernatant from IAM pathway-engineered strains.

Effect of Engineered Strain Supernatant on Root Growth of Water Spinach and Soybean Seeds Under Petroleum Stress

Objective and Methods

To assess the impact of engineered strain supernatant on root growth under petroleum stress, healthy seeds of water spinach and soybeans were selected. Six seeds of each type were randomly divided into two groups: the experimental group (3 seeds) and the control group (3 seeds). The experimental group was treated with the supernatant from an IAM pathway-engineered strain. The engineered strain was cultured overnight in LB medium, and the next day, 1 mL of bacterial culture was transferred to 50 mL of fresh LB medium wrapped in aluminum foil. After 12 hours of incubation at 37°C and 180 rpm, the culture was centrifuged, and the supernatant was collected. Seeds in the experimental group were soaked in the supernatant for 12 hours, while the control group seeds were soaked in water. After soaking, the seeds were transferred to soil containing trace amounts of petroleum and root growth was observed.

Figure 8. Root Growth Under Petroleum Stress with Supernatant from IAM Pathway Strain

Results and Conclusion

The results showed that the root growth in the experimental group was significantly better than in the control group. This indicates that under petroleum stress, seeds treated with the supernatant from the IAA pathway-engineered strain exhibited stronger root growth. As a plant hormone, IAA promotes root development and enhances the plant's ability to adapt to adverse environmental conditions. Therefore, this treatment could potentially improve plant growth in polluted soils, facilitating root establishment and development in petroleum-contaminated environments. This suggests that IAA-producing engineered strains not only promote early root growth but may also enhance plant survival and growth under stress conditions, offering potential applications in environmental remediation.v

Potential Application Directions:

1.Agriculture:

The IAA-producing strain can be used to promote plant growth by enhancing root development and improving seed germination. This is particularly useful for increasing crop yields and improving plant growth in suboptimal soils.

2.Environmental Remediation:

Engineered strains can help in bioremediation of contaminated soils by promoting plant root growth in polluted environments, such as petroleum-contaminated areas. This supports the use of plants in reclaiming polluted land.

3.Stress Resilience:

The part can be applied to increase plant resilience to environmental stresses like drought or soil salinity by boosting root growth and nutrient uptake, helping crops adapt to challenging conditions.

4.Sustainable Agriculture:

Using IAA-producing bacteria as biofertilizers offers a natural, sustainable alternative to chemical fertilizers, improving soil health and supporting ecological farming practices.

References：

1.Spaepen, S., Vanderleyden, J., & Remans, R. (2007). Indole-3-acetic acid in microbial and microorganism-plant signaling. FEMS Microbiology Reviews, 31(4), 425–448.https://doi.org/10.1111/j.1574-6976.2007.00072.x

2.Woodward, A. W., & Bartel, B. (2005). Auxin: Regulation, action, and interaction. Annals of Botany, 95(5), 707–735.https://doi.org/10.1093/aob/mci083

3.Tsavkelova, E. A., Klimova, S. Y., Cherdyntseva, T. A., & Netrusov, A. I. (2006). Microbial producers of plant growth stimulators and their practical use: A review. Applied Biochemistry and Microbiology, 42(2), 117–126.https://doi.org/10.1134/S0003683806020013

4.Patten, C. L., & Glick, B. R. (2002). Role of Pseudomonas putida indoleacetic acid in the development of the host plant root system. Applied and Environmental Microbiology, 68(8), 3795–3801.https://doi.org/10.1128/AEM.68.8.3795-3801.2002

5.Friml J. Auxin transport - shaping the plant. Curr Opin Plant Biol. 2003 Feb;6(1):7-12. doi: 10.1016/s1369526602000031. PMID: 12495745.

6.Xie X, Zhang H, Paré PW. Sustained growth promotion in Arabidopsis with long-term exposure to the beneficial soil bacterium Bacillus subtilis (GB03). Plant Signal Behav. 2009 Oct;4(10):948-53. doi: 10.4161/psb.4.10.9709. Epub 2009 Oct 28. PMID: 19826235; PMCID: PMC2801358.

Sequence and Features